Methods for increasing mannose content of recombinant proteins

ABSTRACT

The present invention relates to methods of modulating the mannose content of recombinant proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/704,662, filed Mar. 25, 2022, which is a continuation of U.S. Ser.No. 16/654,933, filed Oct. 16, 2019, now U.S. Pat. No. 11,319,568, whichis a continuation of U.S. patent application Ser. No. 15/652,138 filedJul. 17, 2017, now abandoned, which is a continuation of U.S. Ser. No.14/776,404, filed Sep. 14, 2015, now U.S. Pat. No. 9,822,388, which is aNational Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2014/022738, having an international filing dateof Mar. 10, 2014 and published in English, which claims the benefit ofU.S. Provisional Application No. 61/784,639 filed Mar. 14, 2013, each ofthe foregoing is incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

IgG antibodies produced in mammalian cell cultures may contain variedlevels of high mannose (HM) glycoforms such as Mannose5 (Man5), Mannose6(Man6), Mannose7 (Man7), Mannose8 (Man8) and Mannose9 (Man9). Highmannose glycoform content of therapeutic proteins and antibodies is acritical quality attribute that has been found to affect pharmacokineticproperties of certain therapeutic antibodies (Goetze, et al., (2011)Glycobiology 21, 949-59; Yu, et al., (2012) MAbs 4, 475-87).

Glycoforms of an antibody expressed by Chinese hamster ovary (CHO) hostcell are largely determined during cell line generation and cloneselection. However, HM content can also be affected by cell cultureconditions (Pacis, et al., (2011) Biotechnol Bioeng 108, 2348-2358). Itis common in therapeutic antibody industry to seek a desired range of HMcontent for an antibody product due to process changes, scale-up,improvements or the need to match existing antibody quality attributes.So far, methods applied for manipulating HM content of an antibody incell culture include changes in media compositions, osmolality, pH,temperature, etc (Yu, et al., supra, Pacis et al., supra, Chee FurngWong et al., (2005) Biotechnol Bioeng 89, 164-177; Ahn, et al., (2008)Biotechnol Bioeng 101, 1234-44). The effectiveness of these methods isspecific to cell lines, molecule types and media environment.Additionally these methods tend to also alter antibody productivity,cell culture behavior and other antibody quality attributes. Theeffectiveness of these methods is obtained empirically.

Therefore, there is a need for a method to modulate the high mannoseglycoform content of therapeutic proteins and antibodies. The inventionprovides a method for increasing the high mannose glycoform contentthrough limited glucose in combination with an alternative carbonsource.

SUMMARY OF THE INVENTION

The invention provides a method for modulating one or more high mannoseglycan species on a recombinant protein during a mammalian cell cultureprocess comprising limiting the amount of glucose in the cell culturemedium and supplementing the cell culture medium with galactose orsucrose.

In one embodiment the glucose concentration in the cell culture mediumis sufficient to result in a concentration of glucose in the spentmedium at or about 0 g/L.

In one embodiment the concentration of glucose in the cell culturemedium is from 0 to 8 g/L. In related embodiments the concentration ofglucose in the cell culture medium is from 4 to 6 g/L; 1 to 3 g/L; 2 to3 g/L; 2.5 g/L or 0 g/L.

In one embodiment the concentration of galactose in the cell culturemedium is from 10 to 20 g/L. In related embodiments the concentration ofgalactose is from 10 to 15 g/L; 10 to 12 g/L or 11.5 g/L.

In one embodiment the concentration of sucrose in the cell culturemedium is from 1 to 48 g/L. In a related embodiment the concentration ofsucrose in the cell culture medium is from 16 to 24 g/L.

In one embodiment the limiting amount of glucose is added during aproduction phase.

In one embodiment the high mannose glycan species is mannose 5.

In one embodiment the cell culture process is a perfusion process.

The invention also provides a method for modulating one or more highmannose glycan species on a recombinant protein during mammalian cellculture comprising; establishing a mammalian cell culture in abioreactor with a serum-free defined culture medium containing 5-8 g/Lglucose; growing the mammalian cells during a growth phase andsupplementing the culture medium with bolus feeds of a serum-freedefined feed medium having from 5-8 g/L glucose; initiating a productionphase in the cell culture by perfusion with a serum-free perfusionmedium having 5-15 g/L glucose; at a predetermined time point, perfusingthe cell culture with a low glucose perfusion medium containing orsupplemented with a decreased amount of glucose, wherein said perfusionmedium further contains or is supplemented with galactose.

In one embodiment the decreased amount of glucose is sufficient toresult in a concentration of glucose in the spent medium of at or about0 g/L.

In one embodiment the concentration of the decreased amount of glucosein the low glucose perfusion medium is from 0 to 3 g/L. In relatedembodiments the concentration of the decreased amount of glucose in thelow glucose perfusion medium is from 2 to 3 g/L; 2.5 g/L or 0 g/L.

In one embodiment the concentration of galactose in the perfusion mediumis from 10 to 20 g/L. In related embodiments the concentration ofgalactose in the low glucose perfusion medium is from 10 to 15 g/L; 10to 12 g/L or 11.5 g/L.

In one embodiment perfusion begins on or about day 5 to on or about day9 of the cell culture. In a related embodiment perfusion begins on orabout day 5 to on or about day 7 of the cell culture. In another relatedembodiment perfusion begins when the cells have reached a productionphase.

In another embodiment perfusion comprises continuous perfusion. In arelated embodiment the rate of perfusion is constant.

In one embodiment perfusion is performed at a rate of less than or equalto 1.0 working volumes per day. In a related embodiment perfusion isperformed at a rate that increases during the production phase from 0.25working volume per day to 1.0 working volume per day during the cellculture. In another related embodiment perfusion is performed at a ratethat reaches 1.0 working volume per day on day 9 to day 11 of the cellculture. In another related embodiment perfusion is performed at a ratethat reaches 1.0 working volume per day on day 10 of the cell culture.

In one embodiment the bolus feeds of serum-free feed medium begin on day3 or day 4 of the cell culture.

In one embodiment the mammalian cell culture is established byinoculating the bioreactor with at least 0.5×10⁶ to 3.0×10⁶ cells/mL ina serum-free culture medium. In a related embodiment the mammalian cellculture is established by inoculating the bioreactor with at least0.5×10⁶ to 1.5×10⁶ cells/mL in a serum-free culture medium.

In one embodiment the high mannose glycan species is Mannose 5.

In one embodiment the method described above further comprisestemperature shift from 36° C. to 31° C.

In one embodiment the method described above further comprises atemperature shift from 36° C. to 33° C. In a related embodiment thetemperature shift occurs at the transition between the growth phase andproduction phase. In a related embodiment the temperature shift occursduring the production phase.

In one embodiment the method above further comprising inducing cellgrowth-arrest by L-asparagine starvation followed by perfusion with aserum-free perfusion medium having an L-asparagine concentration of 5 mMor less. In a related embodiment the concentration of L-asparagine inthe serum-free perfusion medium is less than or equal to 5 mM. In arelated embodiment the concentration of L-asparagine in the serum-freeperfusion medium is less than or equal to 4.0 mM. In another relatedembodiment the concentration of L-asparagine in the serum-free perfusionmedium is less than or equal to 3.0 mM. In another related embodimentthe concentration of L-asparagine in the serum-free perfusion medium isless than or equal to 2.0 mM. In another related embodiment theconcentration of L-asparagine in the serum-free perfusion medium is lessthan or equal to 1.0 mM. In yet another related embodiment theconcentration of L-asparagine in the serum-free perfusion medium is 0mM. In yet another related embodiment the L-asparagine concentration ofthe cell culture medium is monitored prior to and during L-asparaginestarvation.

In one embodiment the method above, further comprises that the packedcell volume during a production phase is less than or equal to 35%. In arelated embodiment the packed cell volume is less than or equal to 35%.In a related embodiment the packed cell volume is less than or equal to30%.

In one embodiment the viable cell density of the mammalian cell cultureat a packed cell volume less than or equal to 35% is 10×10⁶ viablecells/ml to 80×10⁶ viable cells/ml. In a related embodiment the viablecell density of the mammalian cell culture is 20×10⁶ viable cells/ml to30×10⁶ viable cells/ml.

In one embodiment the perfusion is accomplished by alternatingtangential flow. In a related embodiment the perfusion is accomplishedby alternating tangential flow using an ultrafilter or a microfilter.

In one embodiment the bioreactor has a capacity of at least 500 L. In arelated embodiment the bioreactor has a capacity of at least 500 L to2000 L. In a related embodiment the bioreactor has a capacity of atleast 1000 L to 2000 L.

In one embodiment the mammalian cells are Chinese Hamster Ovary (CHO)cells. In one embodiment the recombinant protein is selected from thegroup consisting of a human antibody, a humanized antibody, a chimericantibody, a recombinant fusion protein, or a cytokine.

In one embodiment the method above further comprises a step ofharvesting the recombinant protein produced by the cell culture.

In one embodiment the recombinant protein produced by the cell cultureis purified and formulated in a pharmaceutically acceptable formulation.

In one embodiment the recombinant protein production in the high mannoseglycan species are increased compared to a culture where the cells arenot subjected to limited glucose in combination with galactose.

In one embodiment the concentration of the perfusion medium is 15 g/L.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F show cell culture and Man5 profiles in a fed-batch process.FIG. 1A shows glucose concentration g/L in culture supernatant. FIG. 1Bshows galactose concentration g/L in culture supernatant. FIG. 1C showsViable Cell Density. FIG. 1D shows Viability. FIG. 1E shows Titer. FIG.1F shows Man5. Glucose 1 g/L, galactose 0 g/L (open triangle). Glucose 1g/L, galactose 4 g/L (solid triangle). Glucose 2 g/L, galactose 0 g/L(open circle). Glucose 2 g/L, galactose 4 g/L (solid triangle). Glucose3 g/L, galactose 0 g/L (open square). Glucose 3 g/L, galactose 4 g/L(solid square).

FIGS. 2A-2F show cell culture and amino acid profiles in perfusionprocess. FIG. 2A shows Viable Cell Density, FIG. 2B shows Viability,FIG. 2C shows Gln (glutamine) concentration g/L in spent media analysis,FIG. 2D shows Packed Cell Volume Adjusted Titer, FIG. 2E shows Glc(glucose) concentration g/L in spent media analysis, FIG. 2F showsgalactose concentration g/L in spent media analysis. Glucose 2 g/L,galactose 6 g/L and glutamine 10 mM (solid triangle). Glucose 4 g/L,galactose 6 g/L and glutamine 10 mM (solid circle). Glucose 4 g/L,galactose 6 g/L and glutamine 5 mM (open circle).

FIGS. 3A-3G show cell culture profiles in perfusion process. FIG. 3Ashows Glu (glucose) concentration g/L in spent media analysis, FIG. 3Bshows Gal (galactose) concentration g/L in spent media analysis, FIG. 3Cshows lactate concentration, FIG. 3D shows ammonia concentration, FIG.3E shows Viable cell density, FIG. 3F shows Viability, FIG. 3G showsPacked cell volume adjusted titer. Glucose 3 g/L, galactose 13 g/L(solid square). Glucose 0 g/L, galactose 10 g/L (open circle). Glucose 0g/L, galactose 13 g/L (solid circle). Glucose 1.5 g/L, galactose 11.5g/L (star). Glucose 3 g/L, galactose 10 g/L, (open square).

FIGS. 4A-4B show JMP statistical analysis of perfusion process. FIG. 4Ashows Packed Cell Volume Adjusted Titer, FIG. 4B shows Man5.

FIG. 5 shows time course data showing increase in percent of Man5species. −0 g/L glucose 10 g/L galactose; −+0 g/L glucose 13 g/Lgalactose; +1 3 g/L glucose 10 g/L galactose; ++3 g/L glucose 13 g/Lgalactose; OO 1.5 g/L glucose 11.5 galactose.

FIGS. 6A-6G show cell culture profiles in perfusion process. FIG. 6Ashows Glu (glucose) concentration g/L in spent media analysis, FIG. 6Bshows Sucrose concentration g/L in spent media analysis, FIG. 6C showsViable Cell Density, FIG. 6D shows Lactate concentration g/L, FIG. 6Eshows Ammonium concentration mM, FIG. 6F shows Viability, FIG. 6G showsPacked Cell Volume Adjusted titer. Glucose 6 g/L, sucrose 24 g/L (solidsquare). Glucose 2 g/L, sucrose 16 g/L (open square). Glucose 2 g/L,sucrose 24 g/L (solid circle). Glucose 4 g/L, sucrose 20 g/L (star).Glucose 2 g/L, sucrose 16 g/L (open circle).

FIGS. 7A-7C show JMP statistical analysis of perfusion process. FIG. 7Ashows Packed Cell Volume Adjusted Titer, FIG. 7B shows Man5, FIG. 7Cshows Viability.

DETAILED DESCRIPTION OF THE INVENTION

Production of consistent and reproducible recombinant glycoproteinglycoform profiles remains a considerable challenge to thebiopharmaceutical industry. Variations in cell culture processes play asignificant role in antibody glycosylation profiles. Potentialvariability in the cell culture process physicochemical environmentincluding pH, temperature, cell culture media composition, raw materiallot-to-lot variation, medium filtration material, bioreactor scaledifference, gassing strategy (air, oxygen, carbon dioxide) are just afew examples that can potentially alter glycosylation profiles.

It was observed that under conditions of low or limited glucose, thehigh mannose glycoform content of the recombinant protein increased,however, attributes of the cell culture, such as volumetricproductivity, cell viability, and/or density, diminished. Increasing theglucose concentration improved the culture attributes, but decreased thehigh mannose glycoform content.

The invention provides a method for increasing high mannose glycoforms,in particular, Mannose5 (Man5), to achieve desired product qualityattributes while maintaining desirable levels of certain cell cultureparameters such as volumetric productivity, cell viability, and/ordensity, through the use of low or limited concentrations of glucose incombination with an alternate carbon source, in particular, galactose orsucrose. As described herein, culturing cells in a cell culture mediumwhere glucose is limited by lowering the concentration of glucose in thecell culture medium, in combination with an alternative carbon source,resulted in a recombinant protein having am increased concentration ofhigh mannose glucoforms, while maintaining cell growth, viability andtiter at acceptable levels.

During the production phase of a cell culture, desirable cultureparameters, such as viable cell density, cell viability, percent packedcell volume, titer and/or packed cell volume adjusted titer can beestablished by feeding the cell culture a cell culture medium containingsufficient glucose (from 5 g/L to 15 g/L or more) to establish andmaintain these parameters. At such time during the cell cultureproduction run, when it is desirable to increase the high mannoseglycoform content of the recombinant protein being produced, the cellculture is then fed with a cell culture medium wherein the concentrationof glucose is reduced such that will result in the desired increase inhigh mannose content. Such a cell culture medium is characterized by alower concentration of glucose (0-8 g/L) in combination an alternativecarbon source, such as galactose or sucrose.

Factors that determine the degree to which the glucose concentrationwill need to be lowered include which alternate carbon source used andhow much is used; the cell culture production process; the cell type andmass and the glucose consumption. The greater the cell mass in thebioreactor, the greater the glucose consumption by the cell culture andhence the greater the amount of glucose that can be fed while stillmaintaining a limited glucose state that will produce the desiredincrease Man5 glycoform concentration. The manner in which the glucoseis fed to the cell culture can also influence the amount of glucosenecessary to maintain a limited glucose state that will produce thedesired increase Man5 glycoform concentration. For example, in afed-batch cell culture, glucose can be formulated into the cell culturemedium and supplemented by bolus feeds. In a perfusion cell cultureprocess, glucose concentration will depend on the feed rate (g/L/day) ofthe perfusion medium. Examples of both are provided herein. In addition,the amount of glucose in the culture medium during production can bemeasured, such as by spent media analysis for perfusion cultures. It wasobserved that Man5 levels increased when the amount of glucose in thespent medium was at or nearly 0 g/L.

High mannose glycoform production was increased when situations whereglucose concentrations were decreased. However, low levels of glucosecan impact the production of recombinant proteins in cell culturesystems. Volumetric production, cell viability and viable cell densitycan all be negatively impacted in situations when glucose is limited. Itwas found that the addition of an alternate carbon source, such asgalactose, to cell culture during a period of low or limited glucose wasnot slowed or stabilized the decreases in volumetric production, cellviability and viable cell density, while preserving the increased Man5glycoforms. Alternatively, during a period of low or limited glucose,sucrose was also able to promote high mannose glycoform production,freeing some glucose to maintain volumetric production, cell viabilityand viable cell density. While cells could consume galactose, they didnot consume sucrose in a limited glucose situation. It is believed thatsucrose has an osmolality-related effect on cell metabolism andglycosylation of the molecule. Having the ability to manipulate andmaintain the high mannose glycoform content of a recombinant proteinduring cell culture while minimizing product titer loss and maintainingcell viability represents a valuable and easily-implemented method forcommercial therapeutic protein production.

Provided herein is a method of culturing mammalian cells that is usefulfor increasing high mannose glycoforms, in particular, Man 5, to achievedesired product quality attributes while maintaining acceptable producttiter and cell viability by making use of a limiting amount of glucosein combination with an alternate carbon source, in particular, galactoseor sucrose. The method provides culturing mammalian cells during growthand/or production phases in a cell culture medium having a high,non-limiting glucose concentration, from 5 to 15 g/L glucose, eithercompounded into the medium formulation, supplemented through bolus orcontinuous feeds or both. When viable cell density, cell viabilityand/or titer reach desired levels, the amount of glucose in the cellculture medium is lowered to a limiting amount, such that in theperfusion medium feed for example, the amount of glucose measured inspent medium is at or just above 0 g/L. The rate of glucose consumptionis determined by the rate of glucose addition and/or the mass of thecell culture. Glucose can be fed at up to 8 g/L. In one embodiment,glucose is fed up to 6 g/L. In another embodiment glucose is fed up to 4g/L. In another embodiment, glucose is fed up to 3 g/L. In anotherembodiment glucose is fed up to 2-3 g/L. In yet another embodimentglucose is fed up to 2.5 g/L. In another embodiment, glucose is 0 g/L.

In combination with the lowered glucose concentration, the cell culturemedium contains or is supplemented with galactose, at a concentration upto 20 g/L. In one embodiment the concentration of galactose is from 10to 15 g/L. In another embodiment the concentration of galactose is 11.5g/L.

In another embodiment, in combination with the lowered glucoseconcentration, the cell culture medium contains or is supplemented withsucrose, at a concentration up to 48 g/L. In one embodiment theconcentration of sucrose is 16 to 24 g/L.

Carbohydrate moieties are described herein with reference to commonlyused nomenclature for oligosaccharides. A review of carbohydratechemistry which uses this nomenclature can be found, for example, inHubbard and Ivatt, Ann. Rev. Biochem. 50:555-583 (1981). Thisnomenclature includes, for instance, Man, which represents mannose; Galwhich represents galactose; and Glc, which represents glucose.

By “cell culture” or “culture” is meant the growth and propagation ofcells outside of a multicellular organism or tissue. Suitable cultureconditions for mammalian cells are known in the art. See e.g. Animalcell culture: A Practical Approach, D. Rickwood, ed., Oxford UniversityPress, New York (1992). Mammalian cells may be cultured in suspension orwhile attached to a solid substrate. Fluidized bed bioreactors, hollowfiber bioreactors, roller bottles, shake flasks, or stirred tankbioreactors, with or without microcarriers, can be used.

The mammalian cell culture is grown in a bioreactor. In one embodiment500 L to 20000 L bioreactors are used. In a preferred embodiment, 1000 Lto 2000 L bioreactors are used.

The bioreactor is inoculated with at least 0.5×10⁶ up to and beyond3.0×10⁶ viable cells/mL in a serum-free culture medium. In a preferredembodiment the inoculation is 1.0×10⁶ viable cells/mL.

Once inoculated into the production bioreactor the mammalian cellsundergo an exponential growth phase. The growth phase can be maintainedusing a fed-batch process with bolus feeds of a serum-free feed mediumhaving from 5 to 8 g/L glucose. These supplemental bolus feeds typicallybegin shortly after the cells are inoculated into the bioreactor, at atime when it is anticipated or determined that the cell culture needsfeeding. For example, supplemental feeds can begin on or about day 3 or4 of the culture or a day or two earlier or later. The culture mayreceive two, three, or more bolus feeds during the growth phase. Neitherthe basal cell culture medium nor the bolus feed medium containgalactose or sucrose.

When the cells enter the stationary or production phase, or the cellculture has achieved a desired viable cell density and/or cell titer,the fed batch bolus feeds are discontinued and perfusion is started.Perfusion culture is one in which the cell culture receives freshperfusion feed medium while simultaneously removing spent medium.Perfusion can be continuous, step-wise, intermittent, or a combinationof any or all of any of these. Perfusion rates can be less than aworking volume to many working volumes per day. Preferably the cells areretained in the culture and the spent medium that is removed issubstantially free of cells or has significantly fewer cells than theculture. Perfusion can be accomplished by a number of means includingcentrifugation, sedimentation, or filtration, See e.g. Voisard et al.,(2003), Biotechnology and Bioengineering 82:751-65. A preferredfiltration method is alternating tangential flow filtration. Alternatingtangential flow is maintained by pumping medium through hollow-fiberfilter modules. See e.g. U.S. Pat. No. 6,544,424. The hollow-fibermodules can be microfilters or ultrafilters.

When the fed-batch culture reaches a predetermined trigger point, suchas desired cell viability, cell density, percent packed cell volume,titer, packed cell volume adjusted titer, age or the like, a switchbetween fed-batch and perfusion can take place. For example, this switchcan take place on or about day 7 of the culture, but may take place aday or two earlier or later. The perfusion feed formulation containsglucose at a concentration of up to 15 g/L or more, but does not containgalactose or sucrose. In one embodiment, the perfusion medium contains15 g/L glucose.

When the perfusion culture reaches a predetermined trigger point, suchas desired cell viability, cell density, percent packed cell volume,titer, packed cell volume adjusted titer, age or the like, the glucoseconcentration in the cell culture medium is lowered. For example, thisshift may be initiated on day 11 of the culture, but may take place aday or two earlier or later. At that time the cell culture is perfusedwith cell culture medium containing a lower concentration of glucose.Such a lower concentration of glucose will result in a lowerconcentration of glucose measured in the spent media of at or nearly 0g/L. Glucose can be feed at up to 8 g/L. In one embodiment, glucose isfed up to 6 g/L. In another embodiment glucose is fed up to 4 g/L. Inanother embodiment, glucose is fed up to 3 g/L. In another embodimentglucose is 2-3 g/L. In yet another embodiment, glucose is 2.5 g/L. Inanother embodiment, glucose is 0 g/L.

The limited glucose state in the cell culture is maintained bymonitoring the concentration of glucose in the cell culture, such as bymeasuring glucose concentration in the spent medium, and adjusting theglucose concentration in the perfusion medium formulation to maintain alevel of at or nearly 0 g/L in the spent medium.

The cell culture medium containing the lower concentration of glucosemay also be supplemented with galactose at a concentration of up to 20g/L. In one embodiment the concentration of galactose is from 10 to 15g/L. In another embodiment the concentration of galactose is 11.5 g/L.

Alternatively, the lower glucose cell culture medium may be supplementedwith sucrose at a concentration of 1 to 48 g/L. In one embodiment thesucrose concentration is 16 to 24 g/L.

The cell culture can be continuously maintained in a limited glucosestate supplemented with galactose or sucrose. The cell culture can bemaintained in a limited glucose state supplemented with galactose orsucrose until harvest. The cell culture can be restored to a non-glucoselimited state without galactose or sucrose supplements and the entireprocess begun again.

The cell culture could also be maintained in a perfusion culture systemfor both the growth and production phases. Once inoculated into theproduction bioreactor the mammalian cells undergo an exponential growthphase during which time the cell culture is perfused with serum-freeand/or chemically defined cell culture medium supplemented with 5 to 15g/L glucose. The cell culture medium does not contain galactose orsucrose. The culture is maintained until a desired trigger point isachieved, for example desired viable cell density, cell viability,percent packed cell volume, titer, packed cell adjusted volume titer,age or the like. At that time the cell culture is perfused with a cellculture medium containing a limiting concentration of glucose. Such alimiting concentration of glucose will result in a concentration ofglucose in the spent media of at or nearly 0 g/L glucose. Glucose can befeed at up to 8 g/L. In one embodiment, glucose is fed up to 6 g/L. Inanother embodiment glucose is fed up to 4 g/L. In another embodiment,glucose is fed up to 3 g/L. In another embodiment glucose is 2-3 g/L. Inyet another embodiment, glucose is 2.5 g/L. In another embodiment,glucose is 0 g/L.

The cell culture medium containing the limiting amount of glucose mayalso contain galactose at a concentration of up to 20 g/L. In oneembodiment the concentration of galactose is from 10 to 15 g/L. Inanother embodiment the concentration of galactose is 11.5 g/L.

Alternatively, the cell culture medium containing the limiting amount ofglucose may contain sucrose at a concentration from 1 to 48 g/L. Oneembodiment of the sucrose concentration is 16 to 24 g/L.

In addition, the cell culture medium containing the limiting amount ofglucose may also contain glutamine in addition to galactose or sucrose.Glutamine is at a concentration of 1 to 20 mM in combination with eithergalactose or sucrose. In one embodiment the concentration of glutamineis from 5 to 10 mM.

Viable cell density may be a signal for transition to the productionphase or to lower the glucose concentration in the cell culture medium.It may also be desirable to maintain a certain range or level of viablecell density during the production phase. In one embodiment the viablecell density is 10×10⁶ viable cells/mL to at least about 60×10⁶ viablecells/mL. In another embodiment the viable cell density is 10×10⁶ viablecells/mL to 50×10⁶ viable cells/mL. In another embodiment the viablecell density is 10×10⁶ viable cells/mL to 40×10⁶ viable cells/mL. In apreferred embodiment the viable cell density is 10×10⁶ viable cells/mLto 30×10⁶ viable cells/mL. In another preferred embodiment the viablecell density is 10×10⁶ viable cells/mL to 20×10⁶ viable cells/mL. Inanother preferred embodiment the viable cell density is 20×10⁶ viablecells/mL to 30×10⁶ viable cells/mL. In yet another preferred embodimentthe viable cell density is 20×10⁶ viable cells/mL to 25×10⁶ viablecells/mL. In an even more preferred embodiment the viable cell densityis at least about 20×10⁶ viable cells/mL.

The percent packed cell volume (% PCV) may also be used as a signal fortransition to the production phase or to begin feeding the cell culturewith a cell culture medium containing a limiting amount of glucose. Thecell culture may also be maintained at a desired packed cell volumeduring the production phase. In one embodiment the packed cell volume isequal to or less than 30%. In a preferred embodiment the packed cellvolume is at least about 15-30%. In a preferred embodiment the packedcell volume is at least about 20-25%. In another preferred embodimentthe packed cell volume is equal to or less than 25%. In anotherpreferred embodiment the packed cell volume is equal to or less than15%. In another preferred embodiment the packed cell volume is equal toor less than 20%. In yet another preferred embodiment the packed cellvolume is equal to or less than 15%.

A perfusion cell culture medium having a reduced concentration ofasparagine can be used to arrest cell growth while maintainingproductivity and viability during the production phase. In a preferredembodiment the concentration of asparagine is at least about 0 mM to atleast about 5 mM asparagine, see WIPO Publication No. WO 2013/006479.

As used herein, “perfusion flow rate” is the amount of media that ispassed through (added and removed) from a bioreactor, typicallyexpressed as some portion or multiple of the working volume, in a giventime. “Working volume” refers to the amount of bioreactor volume usedfor cell culture. In one embodiment the perfusion flow rate is oneworking volume or less per day. Perfusion feed medium can be formulatedto maximize perfusion nutrient concentration to minimize perfusion rate.

As used herein, “cell density” refers to the number of cells in a givenvolume of culture medium. “Viable cell density” refers to the number oflive cells in a given volume of culture medium, as determined bystandard viability assays (such as trypan blue dye exclusion method).

As used herein, “packed cell volume” (PCV), also referred to as “percentpacked cell volume” (% PCV), is the ratio of the volume occupied by thecells, to the total volume of cell culture, expressed as a percentage(see Stettler, wt al., (2006) Biotechnol Bioeng. December20:95(6):1228-33). Packed cell volume is a function of cell density andcell diameter; increases in packed cell volume could arise fromincreases in either cell density or cell diameter or both. Packed cellvolume is a measure of the solid content in the cell culture. Solids areremoved during harvest and downstream purification. More solids meanmore effort to separate the solid material from the desired productduring harvest and downstream purification steps. Also, the desiredproduct can become trapped in the solids and lost during the harvestprocess, resulting in a decreased product yield. Since host cells varyin size and cell cultures also contain dead and dying cells and othercellular debris, packed cell volume is a more accurate way to describethe solid content within a cell culture than cell density or viable celldensity.

For the purposes of this invention, cell culture medium is a mediumsuitable for growth of animal cells, such as mammalian cells, in invitro cell culture. Cell culture media formulations are well known inthe art. Typically, cell culture media are comprised of buffers, salts,carbohydrates, amino acids, vitamins and trace essential elements.“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. Various tissue culture media,including defined culture media, are commercially available, forexample, any one or a combination of the following cell culture mediacan be used: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's ModifiedEagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium,Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5AMedium, Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™300 Series (JRH Biosciences, Lenexa, Kansas), among others. Serum-freeversions of such culture media are also available. Cell culture mediamay be supplemented with additional or increased concentrations ofcomponents such as amino acids, salts, sugars, vitamins, hormones,growth factors, buffers, antibiotics, lipids, trace elements and thelike, depending on the requirements of the cells to be cultured and/orthe desired cell culture parameters.

Cell culture media may be serum-free, protein-free, and/or peptone-free.“Serum-free” applies to a cell culture medium that does not containanimal sera, such as fetal bovine serum. “Protein-free” applies to cellculture media free from exogenously added protein, such as transferrin,protein growth factors IGF-1, or insulin. Protein-free media may or maynot contain peptones. “Peptone-free” applies to cell culture media whichcontains no exogenous protein hydrolysates such as animal and/or plantprotein hydrolysates. Cell culture broth or like terminology refers tothe cell culture media that contains, among other things, viable andnon-viable mammalian cells, cell metabolites and cellular debris such asnucleic acids, proteins and liposomes.

Cell cultures can also be supplemented with concentrated feed mediumcontaining components, such as nutrients and amino acids, which areconsumed during the course of the production phase of the cell culture.Concentrated feed medium may be based on just about any cell culturemedia formulation. Such a concentrated feed medium can contain anywherefrom a single or nearly almost all of the components of the cell culturemedium at, for example, about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×,20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or even about 1000× oftheir normal amount, see for example WIPO Publication No WO2012/145682.

The method according to the present invention may be used to improve theproduction of recombinant proteins in multiple phase culture processes.In a multiple stage process, cells are cultured in two or more distinctphases. For example cells may be cultured first in one or more growthphases, under environmental conditions that maximize cell proliferationand viability, then transferred to a production phase, under conditionsthat maximize protein production. In a commercial process for productionof a protein by mammalian cells, there are commonly multiple, forexample, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases thatoccur in different culture vessels preceding a final production culture.The growth and production phases may be preceded by, or separated by,one or more transition phases. In multiple phase processes, the methodaccording to the present invention can be employed at least during thegrowth and production phase of the final production phase of acommercial cell culture, although it may also be employed in a precedinggrowth phase. A production phase can be conducted at large scale. Alarge scale process can be conducted in a volume of at least about 100,500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters.In a preferred embodiment production is conducted in 500 L, 1000 Land/or 2000 L bioreactors. A growth phase may occur at a highertemperature than a production phase. For example, a growth phase mayoccur at a first temperature from about 35° C. to about 38° C., and aproduction phase may occur at a second temperature from about 29° C. toabout 37° C., optionally from about 30° C. to about 36° C. or from about30° C. to about 34° C. In addition, chemical inducers of proteinproduction, such as, for example, caffeine, butyrate, and hexamethylenebisacetamide (HMBA), may be added at the same time as, before, and/orafter a temperature shift. If inducers are added after a temperatureshift, they can be added from one hour to five days after thetemperature shift, optionally from one to two days after the temperatureshift. The cell cultures can be maintained for days or even weeks whilethe cells produce the desired protein(s).

Typically the cell cultures that precede the final production culture(N-x to N-1) are used to generate the seed cells that will be used toinoculate the production bioreactor, the N-1 culture. The seed celldensity can have a positive impact on the level of recombinant proteinproduced. Product levels tend to increase with increasing seed density.Improvement in titer is tied not only to higher seed density, but islikely to be influenced by the metabolic and cell cycle state of thecells that are placed into production.

Seed cells can be produced by any culture method. A preferred method isa perfusion culture using alternating tangential flow filtration. An N-1bioreactor can be run using alternating tangential flow filtration toprovide cells at high density to inoculate a production bioreactor. TheN-1 stage may be used to grow cells to densities of >90×10⁶ cells/mL.The N-1 bioreactor can be used to generate bolus seed cultures or can beused as a rolling seed stock culture that could be maintained to seedmultiple production bioreactors at high seed cell density. The durationof the growth stage of production can range from 7 to 14 days and can bedesigned so as to maintain cells in exponential growth prior toinoculation of the production bioreactor. Perfusion rates, mediumformulation and timing are optimized to grow cells and deliver them tothe production bioreactor in a state that is most conducive tooptimizing their production. Seed cell densities of >15×10⁶ cells/mL canbe achieved for seeding production bioreactors.

The cell lines (also referred to as “host cells”) used in the inventionare genetically engineered to express a polypeptide of commercial orscientific interest. Cell lines are typically derived from a lineagearising from a primary culture that can be maintained in culture for anunlimited time. Genetically engineering the cell line involvestransfecting, transforming or transducing the cells with a recombinantpolynucleotide molecule, and/or otherwise altering (e.g., by homologousrecombination and gene activation or fusion of a recombinant cell with anon-recombinant cell) so as to cause the host cell to express a desiredrecombinant polypeptide. Methods and vectors for genetically engineeringcells and/or cell lines to express a polypeptide of interest are wellknown to those of skill in the art; for example, various techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring LaboratoryPress, 1989); Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990,pp. 15-69.

Animal cell lines are derived from cells whose progenitors were derivedfrom a multi-cellular animal. One type of animal cell line is amammalian cell line. A wide variety of mammalian cell lines suitable forgrowth in culture are available from the American Type CultureCollection (Manassas, Va.) and commercial vendors. Examples of celllines commonly used in the industry include VERO, BHK, HeLa, CV1(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1),PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells arewidely used for the production of complex recombinant proteins, e.g.cytokines, clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362;McKinnon et al. (1991), J. Mol Endocrinol 6:231-239; Wood et al. (1990),J. Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44, are desirable CHO host cell lines becausethe efficient DHFR selectable and amplifiable gene expression systemallows high level recombinant protein expression in these cells (KaufmanR. J. (1990), Meth Enzymol 185:537-566). In addition, these cells areeasy to manipulate as adherent or suspension cultures and exhibitrelatively good genetic stability. CHO cells and proteins recombinantlyexpressed in them have been extensively characterized and have beenapproved for use in clinical commercial manufacturing by regulatoryagencies.

The methods of the invention can be used to culture cells that expressrecombinant proteins of interest. The expressed recombinant proteins maybe secreted into the culture medium from which they can be recoveredand/or collected. In addition, the proteins can be purified, orpartially purified, from such culture or component (e.g., from culturemedium) using known processes and products available from commercialvendors. The purified proteins can then be “formulated”, meaning bufferexchanged, sterilized, bulk-packaged, and/or packaged for a final user.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 18th ed. 1995, MackPublishing Company, Easton, Pa.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. “Glycoprotein” refers to peptides, polypeptidesand proteins, having at least one oligosaccharide side chain includingmannose residues. Glycoproteins may be homologous to the host cell, ormay be heterologous, i.e., foreign, to the host cell being utilized,such as, for example, a human glycoprotein produced by a Chinese hamsterovary (CHO) host-cell. Polypeptides can be of scientific or commercialinterest, including protein-based drugs. Polypeptides include, amongother things, antibodies, fusion proteins, and cytokines. Peptides,polypeptides and proteins are produced by recombinant animal cell linesusing cell culture methods and may be referred to as “recombinantpeptide”, “recombinant polypeptide” and “recombinant protein”. Theexpressed protein(s) may be produced intracellularly or secreted intothe culture medium from which it can be recovered and/or collected.

Examples of polypeptides that can be produced with the methods of theinvention include proteins comprising amino acid sequences identical toor substantially similar to all or part of one of the followingproteins: tumor necrosis factor (TNF), flt3 ligand (WO 94/28391),erythropoeitin, thrombopoeitin, calcitonin, IL-2, angiopoietin-2(Maisonpierre et al. (1997), Science 277(5322): 55-60), ligand forreceptor activator of NF-kappa B (RANKL, WO 01/36637), tumor necrosisfactor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633),thymic stroma-derived lymphopoietin, granulocyte colony stimulatingfactor, granulocyte-macrophage colony stimulating factor (GM-C SF,Australian Patent No. 588819), mast cell growth factor, stem cell growthfactor (U.S. Pat. No. 6,204,363), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_00682; Rüegg andPytela (1995), Gene 160:257-62) growth hormone, insulin, insulinotropin,insulin-like growth factors, parathyroid hormone, interferons includingα-interferons, γ-interferon, and consensus interferons (U.S. Pat. Nos.4,695,623 and 4,897471), nerve growth factor, brain-derived neurotrophicfactor, synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon,interleukins, colony stimulating factors, lymphotoxin-β, leukemiainhibitory factor, and oncostatin-M. Descriptions of other glycoproteinsmay be found in, for example, Human Cytokines: Handbook for Basic andClinical Research, all volumes (Aggarwal and Gutterman, eds. BlackwellSciences, Cambridge, Mass., 1998); Growth Factors: A Practical Approach(McKay and Leigh, eds., Oxford University Press Inc., New York, 1993);and The Cytokine Handbook, Vols. 1 and 2 (Thompson and Lotze eds.,Academic Press, San Diego, Calif., 2003).

Additionally the methods of the invention would be useful to produceproteins comprising all or part of the amino acid sequence of a receptorfor any of the above-mentioned proteins, an antagonist to such areceptor or any of the above-mentioned proteins, and/or proteinssubstantially similar to such receptors or antagonists. These receptorsand antagonists include: both forms of tumor necrosis factor receptor(TNFR, referred to as p55 and p′75, U.S. Pat. No. 5,395,760 and U.S.Pat. No. 5,610,279), Interleukin-1 (IL-1) receptors (types I and II; EPPatent No. 0460846, U.S. Pat. No. 4,968,607, and U.S. Pat. No.5,767,064,), IL-1 receptor antagonists (U.S. Pat. No. 6,337,072), IL-1antagonists or inhibitors (U.S. Pat. Nos. 5,981,713, 6,096,728, and5,075,222) IL-2 receptors, IL-4 receptors (EP Patent No. 0 367 566 andU.S. Pat. No. 5,856,296), IL-15 receptors, IL-17 receptors, IL-18receptors, Fc receptors, granulocyte-macrophage colony stimulatingfactor receptor, granulocyte colony stimulating factor receptor,receptors for oncostatin-M and leukemia inhibitory factor, receptoractivator of NF-kappa B (RANK, WO 01/36637 and U.S. Pat. No. 6,271,349),osteoprotegerin (U.S. Pat. No. 6,015,938), receptors for TRAIL(including TRAIL receptors 1, 2, 3, and 4), and receptors that comprisedeath domains, such as Fas or Apoptosis-Inducing Receptor (AIR).

Other proteins that can be produced using the invention include proteinscomprising all or part of the amino acid sequences of differentiationantigens (referred to as CD proteins) or their ligands or proteinssubstantially similar to either of these. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the Vlth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD proteins are disclosed in subsequent workshops. Examples ofsuch antigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB and OX40. Theligands are often members of the TNF family, as are 41BB ligand and OX40ligand.

Enzymatically active proteins or their ligands can also be produced bythe invention. Examples include proteins comprising all or part of oneof the following proteins or their ligands or a protein substantiallysimilar to one of these: a disintegrin and metalloproteinase domainfamily members including TNF-alpha Converting Enzyme, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, ligands for any of theabove-mentioned enzymes, and numerous other enzymes and their ligands.

The term “antibody” includes reference to immunoglobulins of any isotypeor subclass or to an antigen-binding region thereof that competes withthe intact antibody for specific binding, unless otherwise specified,including human, humanized, chimeric, multi-specific, monoclonal,polyclonal, and oligomers or antigen binding fragments thereof Alsoincluded are proteins having an antigen binding fragment or region suchas Fab, Fab′, F(ab′)₂, Fv, diabodies, Fd, dAb, maxibodies, single chainantibody molecules, complementarity determining region (CDR) fragments,scFv, diabodies, triabodies, tetrabodies and polypeptides that containat least a portion of an immunoglobulin that is sufficient to conferspecific antigen binding to a target polypeptide. The term “antibody” isinclusive of, but not limited to, those that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those thatrecognize any one or a combination of proteins including, but notlimited to, the above-mentioned proteins and/or the following antigens:CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33,CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β, IL-2,IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF,TGF-β2, TGF-β1, EGF receptor (see U.S. Pat. No. 6,235,883) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC1, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, HER-3, a tumor-associatedglycoprotein TAG-72, the SK-1 antigen, tumor-associated epitopes thatare present in elevated levels in the sera of patients with colon and/orpancreatic cancer, cancer-associated epitopes or proteins expressed onbreast, colon, squamous cell, prostate, pancreatic, lung, and/or kidneycancer cells and/or on melanoma, glioma, or neuroblastoma cells, thenecrotic core of a tumor, integrin alpha 4 beta 7, the integrin VLA-4,B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α,the adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, sclerostin, L-selectin, Respiratory Syncitial Virus, humanimmunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcusmutans, and Staphlycoccus aureus. Specific examples of known antibodieswhich can be produced using the methods of the invention include but arenot limited to adalimumab, bevacizumab, infliximab, abciximab,alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, gemtuzumab ozogamicin, golimumab, ibritumomab tiuxetan,labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

The invention can be used to produce recombinant fusion proteinscomprising, for example, any of the above-mentioned proteins. Forexample, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an immunoglobulin, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Hakansson et al.(1999), Structure7:255-64. Specifically included among such recombinant fusion proteinsare proteins in which a portion of a receptor is fused to an Fc portionof an antibody such as etanercept (a p75 TNFR:Fc), and belatacept(CTLA4:Fc). In another embodiment are antibody-drug conjugates.

While the terminology used in this application is standard within theart, definitions of certain terms are provided herein to assure clarityand definiteness to the meaning of the claims. Units, prefixes, andsymbols may be denoted in their SI accepted form. Numeric ranges recitedherein are inclusive of the numbers defining the range and include andare supportive of each integer within the defined range. Unlessotherwise noted, the terms “a” or “an” are to be construed as meaning“at least one of”. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described. The methods and techniques described hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates (1992), and Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990). All documents, or portions ofdocuments, cited in this application, including but not limited topatents, patent applications, articles, books, and treatises, are herebyexpressly incorporated by reference.

The present invention is not to be limited in scope by the specificembodiments described herein that are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

The following examples demonstrate embodiments and aspects of thedisclosed methods and are not intended to be limiting.

EXAMPLES

The substitution of alternative carbohydrate species for glucose withina bioreactor system for the purposes of manipulating high mannoseglycoform content and overall protein quality is addressed.

Example 1 Fed-Batch Culture with Continuous Glucose Feed and BolusGalactose Feed

The effects of reduced glucose and an alternative carbon source on cellculture growth, titer and product quality, particularly the Man5 levels,was evaluated by testing different glucose and galactose concentrationsusing a fed-batch process. The goal of the experimental was to reducethe amount of glucose available in the cell culture medium, whileproviding a different carbon source through a galactose feed.

Twelve 2L Applikon bioreactors were inoculated with CHO cells expressinga recombinant IgG2 antibody at 20e5 viable cells/mL in a working volumeof 1L of a serum free cell culture medium. Cultures were maintained at36° C., 30% dissolved oxygen (DO), 290 rpm agitation, and pH of 6.95. Atyrosine-cystine supplement was fed on days 2 and 5, volumetrically at0.36% based on the initial volume. CO₂ and 1M sodium carbonate base wereadded as needed for pH control.

Bolus feeding of culture media was on days 2, 5 at 9, volumetricallybased on 9% of the initial volume.

A two-factor experiment design was chosen to evaluate cell cultureperformance and product quality attributes with varying amounts ofglucose and galactose in the cell culture medium. The experiment designconsisted of 6 treatments, duplicate bioreactors for each treatment asshown in Table 1. The first factor was continuous glucose feeding(continuous glucose) to deliver 3, 2, or 1 g/day of glucose startingfrom day 2. The treatments with 3 g/day glucose also received additionalbolus glucose feeds to maintain the glucose concentration at 3 g/L. Thepurpose was to ensure that 3 g/day treatments were maintained aspositive controls, never having a glucose limitation.

The second factor was bolus feeds with (1+, 2+, 3+) and without (1−, 2−,3−) galactose (bolus galactose). The objective was to maintain theconcentration of galactose in the cell culture media above 4 g/Lstarting on day 2.

TABLE 1 The 2-factor experimental design for the fed-batch experimentContinuous Bolus Run Glucose, g/day Galactose, g/L 719 2 0 720 3 + bolusglu 0 721 3 + bolus glu 4 722 1 0 723 1 4 724 2 0 725 2 4 726 3 + bolusglu 0 727 2 4 728 1 0 729 3 + bolus glu 4 730 1 4

During the culture run, daily samples were taken to assess the culture.Viable cell density (VCD) and viability were determined bench scaleusing Vi-Cell (Beckman Coulter, Brea, Calif.). Packed cell volume wasdetermined using VoluPAC (Sartorius, Goettingen, Germany). pH, dissolvedcarbon dioxide (pCO2), and dissolved oxygen (pO2) were determined usinga Siemens 248 blood gas analyzer (BGA) (Chiron Diagnostics, CA.Galactose concentration was obtained using a YSI Model 2700 SelectBiochemistry Analyzer (YSI Incorporated, Yellow Springs, Ohio).Metabolite data (glucose, lactate, and ammonia) was obtained usingPolymedco Polychem Analyzer (Polymedco Inc., Cortland Manor, N.Y.).Osmolality was determined using an Advanced Instruments model 2020 microosmometer (Advanced Instruments, Norwood, Mass.). Supernatant sampleswere stored at −80° C. At the end of experiments, frozen cell-freesupernatant samples were thawed and collectively submitted for titer andglycan analysis.

Titer was determined using HPLC analysis. Cell culture supernatantsamples from different time points were thawed and re-filtered in a 96well plate with 0.2 μm membrane. The samples were injected to a HPLCsystem (Hewlett Packard 1100) equipped with UV detection at 280 nm usingPoros® A/20 2.1 mm D ×30 mm L column (Applied Biosystems, Foster City,Calif.) at a flow rate of 2 mL/min. Gradient method using mobile phase100 mM sodium phosphate/250 mM sodium chlorite and 2% Acetic acid/100 mMglycine were used to elute each protein sample for every 5 min.

For glycan analysis, cell culture supernatant samples were collected andpurified by Protein A. The purified samples were treated with PNGase-Fand incubated at 37° C. for 2 hours to release the N-linked glycans. Theenzymatically released glycans were labeled with 2-aminobenzoic acid(2-AA) at 80° C. for 75 minutes. Excess 2-AA label was then removed witha Glycoclean S cartridge. The samples were evaporated overnight and theresulting dry pellet was reconstituted with water for subsequent HILIC(hydrophilic interaction liquid chromatography) analysis. The glycanswere injected and bound to the column in high organic conditions andeluted with an increasing gradient of an aqueous ammonium formatebuffer. Fluorescence detection was used to monitor the glycan elutionand the relative percentage of the major and minor glycan species werecalculated.

Results from Fed-Batch Process with Continuous Glucose Feed and BolusGalactose Feed

The cell culture treatments that received 3 g/day of continuous glucosefeed (Runs 720, 721, 726, and 729), also received bolus glucose feed tokeep their level above 3 g/L. Runs 720 and 726, which received no bolusgalactose feed, routinely required bolus feeds of glucose, while theruns receiving galactose (runs 721 and 729) did not require bolusglucose feeds as often. The cell cultures receiving 1 or 2 g/day ofcontinuous glucose feed, did not receive any additional bolus glucosefeeds. The spent media analysis of these cultures showed the glucoseconcentration was 0 g/L on day 4 regardless of whether or not theculture received bolus galactose feeds. (FIG. 1A).

The cell cultures receiving bolus galactose feeds (Runs 721, 723, 725,727, 729, and 730), maintained galactose levels above 2.5 g/L althoughthe original target was above 4 g/L. (FIG. 1B) When the galactoseconsumption numbers were analyzed, it was found that there was astatistically significant difference in how much galactose the culturesconsumed for the cultures with limited glucose (Runs receiving 2 g/dayor 1 g/day continuous glucose feed) or without limited glucose (Runsreceiving 3 g/day continuous glucose feed plus bolus glucose feed). Thecultures with limited glucose consumed an average of 4.60 grams ofgalactose total, while those without a limitation on glucose consumed anaverage of 3.81 grams.

The continuous glucose feeds and bolus galactose feeds had significantimpact on viable cell density and viability. The cultures with limitedglucose (Runs receiving 2 g/day or 1 g/day continuous glucose feed),along with a bolus galactose feed, maintained good viable cell densityand viability. However, the cultures with limited glucose (Runsreceiving 2 g/day or 1 g/day continuous glucose feed) that did notreceive bolus galactose feeds could not maintain viable cell density andviability once the glucose reached a limitation on day 4. (FIGS. 1C and1D)

The above data indicated that galactose could be used as an alternativecarbon source when glucose was limited in the cell culture medium.Although the cultures with limited glucose (Runs receiving 2 g/day or 1g/day continuous glucose feed), along with bolus galactose feed,maintained good viable cell density and viability, the titer was reducedsignificantly compared to those cultures with no glucose limitation(Runs receiving 3 g/day continuous glucose feed), see FIG. 1E.Statistical analysis showed that the continuous glucose feed level hadthe greatest effect on titer; the bolus galactose feed was alsosignificant, but to a lesser degree.

The Man5 levels in the day 7 samples were reduced in proportion to theincrease of glucose feed from 1 g/day to 3 g/day regardless of whetherthere was a galactose feed. Limited glucose was the only statisticallysignificant factor that resulted in Man5 increase in culture. (FIG. 1F)

Example 2 Perfusion Process with Limited Glucose, Galactose asAlternative Carbon Source and the Addition of Glutamine

The above fed-batch study showed that limiting glucose was the onlyfactor that resulted in Man5 increase; however cultures with limitedglucose could not maintain viable cell density or cell viability. Thealternative carbon source, galactose, did not result in Man5 increase,but was catabolized by the CHO cells and maintained good viable celldensity and cell viability in those cultures where it was added.However, titer was reduced significantly with limited glucose levelseven with alternative carbon source present. Achieving desirable productquality without significant improvement in titer is not commerciallyviable, which is the same case as achieving significant improvement intiter without comparable product quality.

In order to achieve the goals of maintaining or improving titer andachieving desirable product quality, the effects of low glucose and analternative carbon source on cell culture performance and Man5 levelswere tested in a perfusion cell culture. The experiment design consistedof 3 treatments, duplicate bioreactors for each treatment.

On day 0, CHO cells expressing a recombinant IgG2 antibody wereinoculated into 3L production bioreactors at 1×10⁶ viable cells/mL in aworking volume of 1300 ml of a serum-free defined cell culture mediumcontaining 5-8 g/L glucose. Cultures were maintained at 36° C.,dissolved oxygen at 30%, agitation at 400 RPM. The culture was grown inbatch mode for three days. The concentration of glucose in spent mediumanalysis ranged from 1-8 g/L.

On days 3 and 6 the culture received bolus feeds of a concentratedserum-free defined feed media, 8% initial working volume on day 3 and 8%initial working volume on day 6. Bolus glucose feeds were done on days3, 4, 5, 6, 7 to maintain a target concentration of 8 g/L glucose in theculture. Glucose in spent medium analysis ranged from 1-8 g/L.

Perfusion was started on day 7 at a perfusion rate of 0.48 Vol/day.Perfusion was accomplished using an alternating tangential flowperfusion and filtration system (Refine Technologies, Hanover, N.J.)with a 30 kDa hollow fiber filter (GE Healthcare, Uppsala, Sweden). Theserum free defined perfusion medium, pH 7.0, contained 15 g/L glucose.Glucose in spent medium analysis ranged from 3-8 g/L.

On day 11 the switch was made to a serum free, defined perfusion mediumnow containing galactose and having a reduced amount of glucose, seeTable 2a. The concentration of glucose in the perfusion medium wasdecreased to 2 g/L or 4 g/L. Galactose was compounded into the medium at6 g/L. Bolus feeds of a 30% galactose stock solution were used as neededto maintain galactose at a concentration of 4 g/L or above in the cellculture.

For this experiment, the perfusion culture medium also includedglutamine at 5 or 10 mM to determine if glutamine, like glucose, had anyeffect on viable cell density, cell viability, titer and/or Man5 levelsin a situation of glutamine limitation. The literature suggested thatlow glutamine concentrations could have a negative impact on cellculture. The cultures were perfused with this cell culture medium untilharvest on day 17.

TABLE 2a Concentration of glucose, galactose and glutamine in the day 11serum free defined perfusion culture medium (pH 7.0) Run Glucose g/LGalactose g/L Glutamine mM 79 2 6 10 81 2 6 10 82 4 6 10 83 4 6 10 88 46 5 89 4 6 5

Cell culture profiles are shown in FIGS. 2A-2F. The viable cell densityand viability profiles show comparable trends (FIG. 2A and FIG. 2B). Theviability of the low glucose (2 g/L) and the low glutamine (5 mM)conditions showed a downward trend between day 15 and 17. However, theconcentration of glutamine in the spent media analysis indicated thatglutamine was not limited under any of the conditions tested (FIG. 2C)

The surprising finding was that the packed cell volume adjusted Titer(PCV Titer) shown in FIG. 2D was close to the values seen for aperfusion process that was not glucose limited. The titer from theperfusion experiments in this example was much greater that that seen inthe fed-batch process of Example 1 which only yielded a 50% titer. Thesedata indicate that the cells exhibit a different physiological statewhen glucose limited in a perfusion process than they do in a fed batchprocess.

Spent media analysis show that the glucose concentration in culturesupernatants treated with 2 g/L glucose in the perfusion medium reachedzero on day 12 and the glucose concentration in culture supernatantstreated with 4 g/L glucose reached zero on day 13, except for Run 83(FIG. 2E). The spend media analysis confirmed that the cultures were ina limited glucose state.

In general, the concentration of galactose in culture determined byspent media analysis remained above 4 g/L (FIG. 2F).

Table 2b shows that Man5 species increased when the glucoseconcentration in the perfusion medium was lowered to 2 g/L. The timecourse data shows that the Man5 species increased with an increase inculture duration (Day 15 to Day 17). All things being equal, when theconcentration of glucose in the perfusion medium was increased to 4 g/L,the Man5 species decreased accordingly. These results indicate thatlimiting glucose resulted in an increase in Man5 levels and that thelimited state could be initiated by reducing glucose concentration inthe perfusion cell culture medium to 2 g/L or lower and confirming withspent media analysis. However, process variation and cell mass canimpact glucose limitation and the modification in Man5 species. Run 83,showed a higher Man5 value than any other run receiving glucose at 4g/L. In this case Run 83 had a higher viable cell density and packedcell volume adjusted titer (FIG. 2A and FIG. 2D) than any of thecultures receiving 2 g/L glucose (Runs 79 and 81) but like Runs 79 and81, it reached a lower glucose concentration as measured by spent mediumanalysis on day 12. This was earlier than any culture receiving glucoseat 4 g/L (FIG. 2E). Therefore, factors such as cell mass and/or processvariation can impact the glucose feed concentration resulting in alimited glucose situation. In this case where cell density was higher onday 12 (FIG. 2A) leading to a near 0 g/L concentration of glucose in thespent medium, a limited glucose state was initiated even though the cellculture was fed with a high glucose (4 g/L) perfusion feed medium. Onceglucose was limited, Man5 levels increased.

TABLE 2b Man5% at day 15 and 17 following the change in perfusion mediumformulation at day 11 Glucose Galactose Glutamine Day 15 Day 17 Run g/Lg/L mM Man5% Man5% 79 2 6 10 9.29 9.84 81 2 6 10 9.28 9.49 82 4 6 104.64 5.62 83 4 6 10 7.56 8.91 88 4 6 5 5.19 5.65 89 4 6 5 5.09 5.63

Example 3 Perfusion Process with a Combination of Reduced Glucose andHigh Galactose

An experiment was performed with a serum free defined perfusion medium(pH 7.0) formulated with glucose at concentrations of 0, 1.5 or 3 g/L.Galactose was added at 10, 11.5 or 13 g/L, based on the totalconsumption rate of experiment described above. Both glucose andgalactose were compounded into perfusion media so the culture could bemaintained without bolus feeds of either glucose or galactose.Compounding reduced the complexity of the process and improvedconsistency. The experiment was performed as described above; using thesame feeding strategies on days 0 to 10. Table 3 provides thecombinations of perfusion medium formulations used on days 11 through17.

TABLE 3 Experiment design of glucose and galactose in perfusion media.Run Glucose g/L Galactose g/L 103 3 13 104 0 10 105 0 13 106 0 10 1071.5 11.5 108 0 13 109* 3 10 111 3 13 112 3 10 113 1.5 11.5 *Run 109 wasexcluded from the figures due to bioreactor operational failure.

The cell culture profiles in FIGS. 3A-3G show that all cell cultureconditions tested were glucose limited following the day 11 switch (FIG.3A) and the galactose concentrations measured in the spent medium assaywas maintained between 4 to 8 g/L (FIG. 3B) which was proportional tothe galactose concentrations compounded in perfusion medium. The lactatelevel dipped to zero once glucose reached a limitation on day 12 (FIG.3C). The ammonia level increased starting on day 10, before glucosereached a limitation (FIG. 3D). It was very interesting to see thatlowering glucose concentration starting on day 11 resulted in lowergrowth, viability, and titer (FIGS. 3E-3G). These results indicate thatthe limited glucose , not glutamine limitation, causes titer reductionin a perfusion process as well as the fed-batch process in Example 1.Also, since glucose levels of 2-3 g/L, and even up to 4 g/L, resulted inthe increase in Man5 levels, the glucose levels also provided some helpin maintaining higher titer levels.

Statistical analysis using JMP software (JMP Inc. Cary, N.C.) revealedthat glucose concentration was the only statistically significant (pvalue=0.032) factor that impacted titer. The galactose was notstatistically significant factor for titer (FIG. 4A). On the other hand,both glucose and galactose were not statistically significant factorsthat impacted Man5 species, but the interaction between glucose andgalactose was statistically significant (p value=0.0613). (FIG. 4B). Thehigher the galactose concentration the greater the effect of the limitedglucose on Man5 species.

Overall the Man5 levels increased and leveled off when glucose rangedfrom 0 to 3 g/L and decreased when the glucose levels were 4 g/L orgreater. FIG. 5 shows the increase in percent of Man5 species on day 11,13, 15, and 17. For all culture conditions, percent Man5 species startedat about 2% on day 11 and then gradually increased to over 10% on day17. The most significant increase in percent Man5 species was on days 11to 13 when glucose became limited.

Since glucose impacts titer, glucose should be fed at the highestconcentration that will still support an increase in Man5 whilemaintaining cell viability, density and titer at an acceptable level,based on the conditions of the cell culture, such as cell mass, the cellculture process and the alternate carbon source used. For example, for aperfusion cell culture having from 15 to 25×10⁶ cells/ml, glucoseconcentrations of 0-4 g/L in combination with galactose at 10-13 g/L,resulted in an increase of Man5.

Example 4 Perfusion Process with Limited Glucose and Sucrose asAlternative Carbon Source

Sucrose was identified as another carbon source associated with anincrease in Man5 levels. In this experiment, the same culture conditionsas described in Example 3 were used, except on day 11, glucoseconcentrations were 2, 4, and 6 g/L and sucrose was used in place ofgalactose at concentrations of 16, 20, and 24 g/L. Both glucose andsucrose were compounded into perfusion media without additional bolusfeeds of either sugar after day 11, see Table 4.

TABLE 4 Experiment design of glucose and sucrose in perfusion media. RunGlucose g/L Sucrose g/L 153 6 24 164 2 16 155 2 24 156 2 24 157 4 20 1586 16 159* 2 16 160 6 24 162 6 16 163 4 20 *Run 159 was excluded from JMPanalysis due to bioreactor operational failure on day 14

FIG. 6A shows all cell cultures conditions achieved a limited glucosestate on day 12. The sucrose levels determined by spent media assayshowed that sucrose was essentially unchanged from the concentration inthe cell culture medium (FIG. 6B). The 1 g/L difference between theconcentration of sucrose in the perfusion medium and in the spent mediumwas derived from sucrose assay variability and dilution effect. Thisdata indicated that sucrose was not catabolized by CHO cells. Sucrosefunctions as a hyperosmomatic stress in cell culture that could impactprotein glycosylation., see Schmelzer and Miller, Biotechnol. Prog.(2002) 18:346-353; Schmelzer and Miller Biotechnol. Bioeng (2002) 77 (4)February 15; U.S. Pat. No. 8,354,105.

The slight increase in viable cell density, viability and titer resultedfrom the increase in glucose concentration from 2 to 6 g/L in perfusionmedium (FIGS. 6C, 6D and 6F). Lactate levels dipped to zero once theglucose reached limitation on day 12 (FIG. 6G). The ammonia levelincreased starting on day 11 after glucose reached limitation andsucrose was added (FIG. 6E).

The statistical analysis using JMP software (JMP Inc. Cary, N.C.)revealed that both glucose concentration (p value=0.0021) and sucrose (pvalue=0.0823) were statistically significant factors that impacted titer(FIG. 7A). The impact of glucose was most significant (FIG. 7B); theimpact of sucrose on titer could be as a result of osmolality stress(Schmelzer and Miller, supra; U.S. Pat. No. 8,354,105).

Both glucose concentration (p value=0.001) and sucrose (p value=0.0012)were statistically significant factors that impacted viability (FIG.7C). Higher glucose concentrations improved viability while highersucrose concentrations reduced viability. Glucose was the onlystatistically significant factor that impacted Man5 species (pvalue=0.019). Overall the Man5 levels were increased when the glucoseconcentration in the cell culture medium ranged from 2 to 6 g/L.

What is claimed is:
 1. A method of modulating the amount of themannose-5 glycoform of an Immunoglobulin G2 (IgG2) molecule in an IgG2composition, wherein said IgG2 composition is produced by a ChineseHamster Ovary (CHO) cell culture, comprising: establishing a cellculture of CHO cells in a bioreactor, wherein the CHO cell produces anIgG2 molecule; limiting the amount of glucose in the bioreactor, whereinthe concentration of the glucose in the spent medium of the bioreactoris from about 0 to 3 g/L; supplementing the cell culture with a feedingmedium comprising galactose, such that the concentration of galactose inthe resulting spent medium of the bioreactor is above 2.5 g/L;harvesting and purifying the IgG2 composition from the cell culture. 2.The method of claim 1, wherein said IgG2 is denosumab and said IgG2composition is a denosumab composition.
 3. The method of claim 1,wherein said IgG2 is panitumumab and said IgG2 composition is apanitumumab composition.
 4. The method of claim 1, wherein said CHO cellculture is maintained for at least 7 days before the IgG2 composition isharvested.
 5. The method of claim 1, wherein the concentration ofgalactose in the resulting spent medium is from 4 to 8 g/L.
 6. Themethod of claim 1, wherein the concentration of galactose in theresulting spent medium is above 4 g/L.
 7. The method of claim 1,comprising feeding the cell culture with a feeding medium, wherein theglucose concentration in the feeding medium is sufficient to result in aconcentration of glucose in the spent medium at about 0 g/L.
 8. Themethod of claim 1, wherein a feeding medium comprising a limiting amountof glucose that is fed during a production phase.
 9. The method of claim1, wherein said cell culture process comprises a perfusion process. 10.The method of claim 1, wherein said cell culture process comprises abolus feed or continuous feed process.
 11. The method of claim 1,wherein the amount of mannose-5 glycoform of the IgG2 molecule in theIgG2 composition is increased, as compared to that of an IgG2composition obtained from a CHO cell culture where the cells are notsubjected to limited glucose in combination with galactose.
 12. Themethod of claim 2, wherein said denosumab composition is formulated in apharmaceutically acceptable formulation.
 13. The method of claim 3,wherein said panitumumab composition is formulated in a pharmaceuticallyacceptable formulation.
 14. A method of producing an immunoglobulin G2(IgG2) composition by a Chinese Hamster Ovary (CHO) cell culture,comprising: establishing a cell culture of CHO cells in a bioreactor,wherein the CHO cell produces an IgG2 molecule; limiting the amount ofglucose in the bioreactor, wherein the concentration of the glucose inthe spent medium of the bioreactor is from about 0 to 3 g/L;supplementing the cell culture with a feeding medium comprisinggalactose, such that the concentration of galactose in the resultingspent medium of the bioreactor is above 2.5 g/L; harvesting andpurifying the IgG2 composition from the cell culture.
 15. The method ofclaim 14, wherein said IgG2 composition is a denosumab composition andsaid IgG2 molecule is denosumab.
 16. The method of claim 14, whereinsaid IgG2 composition is a panitumumab composition and said IgG2molecule is panitumumab.
 17. The method of claim 14, wherein said CHOcell culture is maintained for at least 7 days before the IgG2composition is harvested.
 18. The method of claim 14, wherein theconcentration of galactose in the resulting spent medium is from 4 to 8g/L.
 19. The method of claim 14, wherein the concentration of galactosein the resulting spent medium is above 4 g/L.
 20. The method of claim14, comprising feeding the cell culture with a feeding medium, whereinthe glucose concentration in the feeding medium is sufficient to resultin a concentration of glucose in the spent medium at about 0 g/L. 21.The method of claim 14, wherein a feeding medium comprising a limitingamount of glucose that is fed during a production phase.
 22. The methodof claim 14, wherein said cell culture process comprises a perfusionprocess.
 23. The method of claim 14, wherein said cell culture processcomprises a bolus feed or continuous feed process.
 24. The method ofclaim 14, wherein the amount of mannose-5 glycoform of the IgG2 moleculein the IgG2 composition is increased, as compared to that of an IgG2composition obtained from a CHO cell culture where the cells are notsubjected to limited glucose in combination with galactose.
 25. Themethod of claim 15, wherein said denosumab composition is formulated ina pharmaceutically acceptable formulation.
 26. The method of claim 16,wherein said panitumumab composition is formulated in a pharmaceuticallyacceptable formulation.
 27. A method of producing an immunoglobulin G2(IgG2) composition by a Chinese Hamster Ovary (CHO) cell culture,wherein said IgG2 is denosumab, comprising: establishing a cell cultureof CHO cells in a bioreactor, wherein the CHO cell produces denosumabmolecule; limiting the amount of glucose in the bioreactor, wherein theconcentration of the glucose in the spent medium of the bioreactor isfrom about 0 to 3 g/L; supplementing the cell culture with a feedingmedium comprising galactose, such that the concentration of galactose inthe resulting spent medium of the bioreactor is above 2.5 g/L;harvesting and purifying the denosumab composition from the cellculture.
 28. The method of claim 27, wherein said denosumab compositionis formulated in a pharmaceutically acceptable formulation.